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Regulation of neural progenitor cell state by ephrin-B

Maintaining a balance between self-renewal and differentiation in neural progenitor cells during development is important to ensure that correct numbers of neural cells are generated. We report that the ephrin-B–PDZ-RGS3 signaling pathway functions to regulate this balance in the developing mammalian cerebral cortex. During cortical neurogenesis, expression of ephrin-B1 and PDZ-RGS3 is specifically seen in progenitor cells and is turned off at the onset of neuronal differentiation. Persistent expression of ephrin-B1 and PDZ-RGS3 prevents differentiation of neural progenitor cells. Blocking RGS-mediated ephrin-B1 signaling in progenitor cells through RNA interference or expression of dominant-negative mutants results in differentiation. Genetic knockout of ephrin-B1 causes early cell cycle exit and leads to a concomitant loss of neural progenitor cells. Our results indicate that ephrin-B function is critical for the maintenance of the neural progenitor cell state and that this role of ephrin-B is mediated by PDZ-RGS3, likely via interacting with the noncanonical G protein signaling pathway, which is essential in neural progenitor asymmetrical cell division.

Introduction
In the developing mammalian cerebral cortex, the majority of projection neurons originate from neural progenitor cells located in the ventricular zone (VZ), which lines the lateral ventricles. Initially, neural progenitor cells proliferate to expand their population. As development proceeds, they progressively enter asymmetrical cell divisions and give rise to neurons and glia. Newborn cortical neurons embark on migration away from the VZ into the cortical plate (CP), where they form highly organized connections within the cortex or with subcortical targets. During development, the balance between self-renewal and differentiation of neural progenitor cells is tightly regulated to ensure that correct numbers of neural cells are generated to form a functional cortex. The molecular basis of this critical developmental regulation is, however, not well understood ( McConnell, 1995 ;Monuki and Walsh, 2001 ;Temple, 2001 ;Gupta et al., 2002 ;Fishell and Kriegstein, 2003 ;Gotz and Huttner, 2005 ;Rakic, 2006 ;Molyneaux et al., 2007 ).
The ephrin/Eph family of molecules is known to function in tissue segmentation, axon guidance, neuronal migration, and dendritic and synaptic modulation during neural development ( Flanagan and Vanderhaeghen, 1998 ;Drescher, 2002 ;Poliakov et al., 2004 ;Davy and Soriano, 2005 ;Egea and Klein, 2007 ;Arvanitis and Davy, 2008 ;Pasquale, 2008 ). Recently, ephrin/ Eph molecules have also been suggested to regulate proliferation, differentiation, and survival of neural progenitor/stem cells ( Conover et al., 2000 ;Aoki et al., 2004 ;Depaepe et al., 2005 ;Holmberg et al., 2005 ;Katakowski et al., 2005 ;Ricard et al., 2006 ). The B subfamily of ephrins and Ephs is prominently associated with neural progenitor/stem cells both in embryonic brains Stuckmann et al., 2001 ) and in the adult subventricular zone (SVZ; Conover et al., 2000 ;Ricard et al., 2006 ). Ephrin-Bs are type I membrane proteins containing one membrane-spanning region and a short cytoplasmic domain. After their original identifi cation as ligands for Eph receptors, ephrin-Bs were found to be capable of " reverse signaling " into M aintaining a balance between self-renewal and differentiation in neural progenitor cells during development is important to ensure that correct numbers of neural cells are generated. We report that the ephrin-B -PDZ-RGS3 signaling pathway functions to regulate this balance in the developing mammalian cerebral cortex. During cortical neurogenesis, expression of ephrin-B1 and PDZ-RGS3 is specifi cally seen in progenitor cells and is turned off at the onset of neuronal differentiation. Persistent expression of ephrin-B1 and PDZ-RGS3 prevents differentiation of neural progenitor cells.
Blocking RGS-mediated ephrin-B1 signaling in progenitor cells through RNA interference or expression of dominant-negative mutants results in differentiation. Genetic knockout of ephrin-B1 causes early cell cycle exit and leads to a concomitant loss of neural progenitor cells. Our results indicate that ephrin-B function is critical for the maintenance of the neural progenitor cell state and that this role of ephrin-B is mediated by PDZ-RGS3, likely via interacting with the noncanonical G protein signaling pathway, which is essential in neural progenitor asymmetrical cell division.

Regulation of neural progenitor cell state by ephrin-B
EphB receptors showed that although EphB1 was not detected, EphB2, 3, and 4 were expressed in the VZ to various degrees ( Fig. 1 D ): EphB2 was uniformly expressed in the radial cortical dimension, whereas EphB3 and 4 were preferentially expressed in the VZ. Using EphB2-Fc and ephrin-B1-Fc (a fusion protein between the extracellular domain of EphB2 or ephrin-B1 and human Fc), we further assessed the functional expression of ephrin-B and EphB in neural progenitor cells in acutely dissociated cultures ( Hartfuss et al., 2001 ). Fc control did not give detectable staining on the dissociated neural progenitor cells (unpublished data). EphB2 and Ephrin-B1 binding sites were both found on the surface of nestin-positive progenitor cells ( Fig. 1 E ). EphB2-Fc was also seen to cause patches of staining, indicative of the clustering of ephrin-B ( Bruckner et al., 1999 ;Himanen et al., 2001 ), on the surface of dissociated neural progenitor cells. Collectively, these expression analyses indicate that ephrin-B1, ephrin-B2, PDZ-RGS3, and some EphB receptors are expressed in the cortical VZ by neural progenitor cells.

PDZ-RGS3 prevents differentiation of neural progenitor cells
To explore the function of ephrin-B signaling in neural progenitor cells, we used in utero electroporation-mediated DNA transfection ( Fukuchi-Shimogori and Grove, 2001 ;Ohtsuka et al., 2001 ;Saito and Nakatsuji, 2001 ;Tabata and Nakajima, 2001 ) to achieve gain of function and loss of function of ephrin-B in the cerebral cortex. This electroporation technique has proven to be useful for in vivo functional analyses of molecular organization of the developing cerebral cortex ( Fukuchi-Shimogori and Grove, 2001 ;Bai et al., 2003 ;Elias et al., 2007 ;Mizutani et al., 2007 ). We fi rst tested gain of function of ephrin-B and PDZ-RGS3 by using bicistronic expression under the control of a ubiquitous promoter (elongation factor 2 promoter). To perturb normal mouse cortical neurogenesis that spans the period from embryonic day (E) 12 to 18, we introduced expression plasmids of Ephrin-B1 and PDZ-RGS3 or the control EGFP into the cortices at E13.5 and analyzed the electroporated brains at later stages between E15.5 and 17.5 (Fig. S1 A, avai lable at http://www.jcb.org/cgi/content/full/jcb.200708091/DC1). Control cells expressing EGFP alone showed a progressive accumulation in the CP from E15.5 to 17.5, which is consistent with normal outward migration of cortical neurons born in the VZ. In contrast, cells expressing EGFP-PDZ-RGS3-IRES-HAephrin-B1 were mostly found in the region surrounding the SVZ. For example, at E17.5, 63% of green cells were found in the area encompassing the VZ, SVZ, and IZ (collectively called hereafter the non-CP area) versus 27% in the control. Immunohistochemistry on cells coexpressing ephrin-B1 and PDZ-RGS3 showed that they were positive for nestin but negative for ␤ -III tubulin (Fig. S1 B) and some of the cells could incorporate BrdU with a short pulse of labeling (Fig. S1 C). These results indicate that prolonged expression of ephrin-B1 and PDZ-RGS3 could block normal differentiation of the affected progenitor cells.
The EF2 promoter mentioned in the previous paragraph could cause an overexpression of ephrin-B1 and PDZ-RGS3 in progenitor cells, which would have both endogenously and exogenously expressed proteins. In this situation, it would be hard the bearing cells ( Holland et al., 1996 ;Bruckner et al., 1997 ). This led to a model for bidirectional signaling in contact-mediated cell -cell communication via Eph -ephrin interaction: ephrin-B can both activate " forward signaling " via the Eph receptor and initiate reverse signaling through its own cytoplasmic domain. In one reverse signaling pathway , signaling of ephrin-B is mediated through PDZ-RGS3, a regulator of G protein signaling (RGS) protein known to function as an inhibitor of G protein signaling ( De Vries et al., 2000 ;Ross and Wilkie, 2000 ;Siderovski and Willard, 2005 ).
In the adult SVZ, ephrin-B1 and ephrin-B2 are expressed by astrocytes, which function as neural stem cells ( Conover et al., 2000 ;Ricard et al., 2006 ). Infusion of an ectodomain of EphB (EphB-Fc) or an ectodomain of ephrin-B (ephrin-B-Fc) into the SVZ leads to proliferation of neural stem cells ( Conover et al., 2000 ). These results indicate that the activation or disruption of ephrin-B/EphB signaling can positively regulate stem cell proliferation in the SVZ. Ablation of ephrin-B3, the third member of the ephrin-B subfamily, results in increased neural stem cell proliferation and cell death in the SVZ ( Ricard et al., 2006 ). This observed effect of ephrin-B3 is probably through an indirect process, as ephrin-B3 was reported to be expressed outside the SVZ where neural stem cells reside ( Ricard et al., 2006 ). Hence, the function of ephrin-B in the neurogenic areas of the embryonic or adult brains remains largely unclear.
In the developing cerebral cortex, ephrin-B1 appears to be selectively expressed in the VZ, associating with neuroepithelial progenitor cells but not young neurons Stuckmann et al., 2001 ). Interestingly, PDZ-RGS3, a downstream signaling mediator of ephrin-B reverse signaling, is coexpressed with ephrin-B1 in the cortical VZ . This suggests that ephrin-B signaling may be important for regulating the balance between self-renewal and differentiation during development. In this study, we present evidence in support of RGS-mediated ephrin-B reverse signaling in maintaining the cortical neural progenitor cells. To investigate a potential role for ephrin-B signaling in the developing cerebral cortex, we began by examining expression patterns in more detail. RNAs of ephrin-B1 and PDZ-RGS3 were found selectively in the VZ during cortical neurogenesis  ). Ephrin-B2 RNA was expressed at a lower level in the VZ and more abundantly in the SVZ region (unpublished data). Ephrin-B3 RNA was not detected in the VZ and SVZ areas (unpublished data). Cortical neural progenitor cells are marked by expression of nestin ( Lendahl et al., 1990 ) and the radial glia marker RC2 ( Fishell and Kriegstein, 2003 ;Gotz and Huttner, 2005 ). We found coexpression in the VZ of ephrin-B proteins with nestin and RC2 ( Fig. 1 A ) or PDZ-RGS3 with nestin ( Fig. 1 B ). Furthermore, pulse labeling of bromodeoxyuridine (BrdU) showed that ephrin-B staining colocalized with BrdU-positive cells in the VZ ( Fig. 1 C ). RNA in situ of several the CP at E16.5 and 17.5. Between the two promoters used in these experiments, there were a smaller percentage of cells expressing DCX-EGFP in the non-CP area (53%; Fig. 2 A ) than that of cells expressing EF-EGFP (92%; Fig. S1 A) at E15.5. This difference was likely because of the different specifi city of the two promoters. DCX-EGFP would be specifi cally enriched in migrating neurons, which might represent the pool of progenitor cells that were differentiating or poised to differentiate at the time of electroporation, whereas EF-EGFP would be expected to be expressed in all transfected progenitor cells (progenitors that were differentiating or were at different stages of cell cycle) and perhaps in other cell types in the VZ area. Nonetheless, use of the two different promoters to coexpress ephrin-B1 and PDZ-RGS3 led to similar outcomes.

Ephrin
To address whether the cells that were stuck in the SVZ were not healthy or had not changed their location because of some generalized inability to migrate, we used a Transwell analysis  to assess their motility. Because neural progenitor cells are basic FGF responsive ( Gage, 2000 ), we tested for basic FGF -induced migration. We found that cells expressing both ephrin-B1 and PDZ-RGS3 showed a similar migration profi le toward basic FGF to the control cells expressing EGFP only ( Fig. 2 B ), indicating that continued expression of ephrin-B1 and PDZ-RGS3 did not render them unable to migrate.
We further assessed the expression of cellular markers in cells carrying both ephrin-B1 and PDZ-RGS3 driven by the to know whether a phenotype was a result of a direct effect of gain of function or simply because of an abnormal expression at the progenitor cell stage. To avoid this situation, we further tested coupling-continued expression of ephrin-B1 and PDZ-RGS3 to the onset of neuronal differentiation to prevent their normal down-regulation during cortical neurogenesis. To this end, ephrin-B1 and PDZ-RGS3, either alone or in combination, were expressed under the control of the promoter of the doublecortin (DCX) gene, an early marker for migrating cortical neurons ( Francis et al., 1999 ;Gleeson et al., 1999 ). The DCX promoter used here was found to direct reporter gene expression, which is consistent with the endogenous DCX gene both in electroporation and in transgenic mice . Control cells expressing EGFP alone showed a progressive outward migration pattern, as was seen in cells carrying EGFP driven by the EF2 promoter ( Fig. 2 A and Fig. S1 A). When ephrin-B1 or PDZ-RGS3 was electroporated alone, the transfected cells exhibited a largely similar migratory pattern to that of the control cells. In contrast, cells carrying EGFP-PDZ-RGS3-IRES-HA-ephrin-B1 showed a signifi cant delay in migration or differentiation: at E15.5, while the control cells were in the intermediate zone (IZ) underneath the CP, cells with continued expression of ephrin-B1 and PDZ-RGS3 appeared to be mostly in the region surrounding the SVZ, with some cells even remaining deep in the VZ. In general, most green cells coexpressing ephrin-B1 and PDZ-RGS3 stayed in the SVZ area at all three stages analyzed, with signifi cantly fewer cells entering translated region of ephrin-B1 gene), specifi cally inhibit the expression of ephrin-B1 without affecting the expression of ephrin-B2 or B3 in transfected cells (Fig. S2, A and C, available at http://www.jcb.org/cgi/content/full/jcb.200708091/DC1) or in cortical neural progenitor cells (Fig. S2, B and D). As a control, we also used a mutant-type shRNA (shRNAB1.3mt) that does not affect the expression of ephrin-B1. All shRNAs were expressed by pNUTS, a mouse U6 promoter -containing plasmid which also carries EGFP under the control of the ubiquitin promoter, and were introduced into E13.5 cerebral cortices through in utero electroporation. Electroporated brains were dissected out and sectioned for analyses at E15.5. Although green cells containing shRNAB1.3mt were mostly found in the VZ at E15.5, many green cells carrying the wild-type shRNAB1.3 had already moved into the CP at that stage ( Fig. 3 A ). In general, there was a trend that more cells affected by the wild-type shRNA moved away from the apical part of the VZ than those cells carrying the mutant-type shRNA at E15.5 ( Fig. 3 A ). Electroporation of shRNAB1-3UTR caused a similar shift of cells away from DCX promoter. Immunohistochemistry revealed that these cells were positive for nestin but negative for ␤ III-tubulin ( Fig. 2 C ). Because DCX expression did not seem to be affected in these cells (unpublished data), these results suggest that coexpression of ephrin-B1 and PDZ-RGS3 under the control of the DCX promoter was able to partially block or reverse the progression of differentiation in the affected cells.
Collectively, the results from experiments using both a ubiquitous promoter and a migrating neuron-specifi c promoter suggest a potential role of PDZ-RGS3 -mediated ephrin-B signaling in promoting the maintenance of neural progenitor cells in the cerebral cortex.

Knockdown of ephrin-B1 causes differentiation of neural progenitor cells
We next used RNAi to specifi cally knock down ephrin-B1 expression in the cortex. The short hairpin RNAs (shRNAs) used in our experiments, shRNAB1.3 (sequence within the coding region of ephrin-B1) or shRNAB1-3UTR (sequence within the 3 Ј un- The up-shift pattern might indicate that the progenitor cells affected by RNAi of ephrin-B1 had differentiated. To address whether knockdown of ephrin-B1 expression had either caused neural progenitor cells to differentiate into the ventricle, and this effect could be suppressed by coexpression of the coding sequence (CDS) of ephrin-B1, which is resistant to the shRNA ( Fig. 3 B and Fig. S2 C), suggesting that the effect of RNAi of ephrin-B1 was not likely because of an off-target effect. To more directly assess the effect of RNAi of ephrin-B1 on cell fate, we looked at the electroporated cells at E14.5, when the earliest migrated cells seen in the CP at E15.5 were presumably still in the VZ area. RNAi of ephrin-B1 led to an outward shift of the affected cells away from the apical side of the VZ, as was also seen at E15.5 ( Fig. 4 A ). Analyses on the BrdU incorporation and the number of phospho -Histone H3 + (p-H3 + ) cells showed that RNAi of ephrin-B1 caused a decrease on both the BrdU labeling index (percentage of short pulse-labeled BrdU + S-phase cells in the total number of green cells; Chenn and Walsh, 2002 ) and the mitotic index (percentage of p-H3 + mitotic cells in the total number of green cells; Li et al., 2003 ;Fig. 4 A ). Cell cycle exit analysis further showed that RNAi of ephrin-B1 caused more cells to exit the cell cycle ( Fig. 4 B ).
To address further whether RNAi of ephrin-B1 could activate the differentiation, we transfected shRNAB1.3 or shRNAB1.3mt in vitro into isolated neural progenitor cells derived from early embryonic cortices ( Johe et al., 1996 ). As shown in Fig. 4 C , compared with the mutant control shRNA, shRNAB1.3 reduced the population of nestin-positive and mitotic (BrdU-positive) cells, with a concomitant increase in the number of cells positive for DCX.
Collectively, the results from the knockdown of ephrin-B1 in vivo and in vitro indicate that ephrin-B1 is critical for maintaining the neural progenitor identity in the developing cerebral cortex.

PDZ-RGS3 -dependent signaling mediates the function of ephrin-B
To further study the effect of blocking ephrin-B function on neural progenitor cells, we sought to alternatively take a dominantnegative approach by expressing mutant ephrin-B or PDZ-RGS3 proteins  ). These mutant proteins could provide an independent method for assessing the observed effect of RNAi of ephrin-B and could also offer insights on the contribution of reverse versus forward signaling. Ephrin-B1 ␦ 4 -EGFP was a deletion mutant of ephrin-B1 with the last four amino acids of the cytoplasmic domain (which constitute the PDZ-binding neurons, which would normally migrate outwards, or alternatively had induced premature migration of progenitor cells into the CP, we performed immunohistochemistry on the green cells that had reached the CP. In the cortices electroporated with shRNAB1.3, green cells in the CP were negative for nestin and pulse-labeled BrdU ( Fig. 3 C ) but were positive for two markers for newborn neurons, ␤ III-tubulin and DCX ( Fig. 3 D ). Immunofl uorescent staining on dissociated cells derived from electroporated brains showed that more cells affected by shRNAB1.3 were negative of nestin but positive for DCX ( Fig. 3 E ), further indicating that RNAi of ephrin-B1 could promote differentiation of the affected progenitor cells.  cortical neurogenesis. Ephrin-B1 is X linked, and previous studies ( Compagni et al., 2003 ;Davy et al., 2004Davy et al., , 2006 showed that ephrin-B1 heterozygotes exhibited phenotypes not observed in homozygotes because of sorting out of ephrin-B1 -positive and -negative cells generated by random X inactivation. We thus analyzed cortical neural progenitor cells both in heterozygous and homozygous ephrin-B1 mutant mice. We fi rst performed anti-nestin immunohistochemistry to look at the morphological appearance of neural progenitor cells ( Fig. 6 A ). In wild-type brains, nestin-positive cells typically extend radially oriented long processes in an orderly manner. In mutant mice (null or het), nestin-positive cells in the brain appeared to have a disrupted radial organization (e.g., an uneven nestin staining pattern in the cerebral cortex) or to have lost the radial organization (e.g., in the ganglion eminence, the processes of progenitor cells were shorter and projected in various directions). This abnormality showed a 90% penetrance in nulls and 65% penetrance in heterozygotes. These results indicate that deleting the ephrin-B1 gene causes defects in neural progenitor cell organization in the developing brain.
We next studied the cell cycle dynamics of the mutant neural progenitor cells. We fi rst analyzed the BrdU labeling index motif) replaced with EGFP. The deletion mutants of PDZ-RGS3 were PDZ-EGFP (the entire sequence C terminal to the PDZ domain was replaced by EGFP) and PDZ-RGS3 ␦ RGS -EGFP (the RGS domain was replaced by EGFP). Similar to the effect of RNAi of ephrin-B1, electroporation-mediated cortical expression of these mutant proteins resulted in an outward shift of the affected cells away from the apical side of the VZ and led to more green cells translocating into the CP ( Fig. 5 ). RNAi of PDZ-RGS3 also led to a similar phenotype (unpublished data). These results further support a role of ephrin-B in the maintenance of neural progenitor cells. The data from dominant-negative inhibition also provide evidence that the function of ephrin-B1 in progenitor cells is dependent on reverse signaling and that PDZ-RGS3 is an important downstream mediator.

Genetic loss of function of ephrin-B1 leads to a loss of neural progenitor cells
The electroporation studies suggest that genetic loss of function of ephrin-B reverse signaling might cause early differentiation and hence a reduction in the number of cortical neural progenitor cells. We therefore assessed neural progenitor cells in ephrin-B1 knockout mice ( Davy et al., 2004 ) during the period of venous and arterial vasculature ( Wang et al., 1998 ;Adams et al., 1999 ), and in the skeletal defects (polydactyly and cranial foramen) observed in the heterozygote females of the ephrin-B1 mutant mice ( Compagni et al., 2003 ;Davy et al., 2004Davy et al., , 2006 were based on EphB -ephrin-B -mediated cell sorting. In contrast, ephrin-B1 could play a cell autonomous role in neural crest cells to control their migration ( Davy et al., 2004 ). Our data of ephrin-B in regulating the self-renewal of cortical neural progenitor cells appear to be also consistent with a cell-autonomous ephrin-B function.
In the postnatal (postnatal day 23) ephrin-B1 knockout mice, the overall brain morphology, including cortex and hippocampus, appeared to be largely comparable among the null and wild-type brains, although the surviving ephrin-B1 nulls were, on average, 40% lighter in body weight compared with wild-type mice, which suggests growth retardation (unpublished data). As cortical VZ/ SVZ expression of ephrin-B2 persisted in the ephrin-B1 -null brains (unpublished data), double knockout of ephrin-B1 and -B2 or knockout of PDZ-RGS3 will need to be further analyzed to alleviate possible functional redundancy in the cortex. It would also be interesting to investigate whether the postnatal cortex might have a similar self-repair plasticity in fi xing early defects as revealed in the adult SVZ .

Signaling of B and A subfamily of ephrins in neural progenitor/stem cells
Our current results lend further support to the recently reported roles of ephrins in neural progenitor/stem cells. In addition, our data reveal functional differences between the two subfamilies of ephrins in neural progenitor cells. Although our study indicates that ephrin-B reverse signaling is critical for the maintenance of cortical neural progenitor cells, transgenic expression of ephrin-A5 in the cortex causes a decrease in the number of neural progenitor cells ( Depaepe et al., 2005 ), suggesting that ephrin-A/EphA signaling (activation of EphA or gain of ephrin-A reverse signaling) negatively regulates the progenitor numbers. In the adult SVZ, infusion of EphB-Fc/ephrin-B-Fc ( Conover et al., 2000 ) or EphA-Fc/ephrin-A-Fc ( Holmberg et al., 2005 ) leads to a similar outcome: more proliferation of neural stem cells, which suggests that either activation of ephrin-B/EphB and ephrin-A/EphA signaling or the disruption of it activates neural stem cell proliferation. Because the knockout of ephrin-A2 or EphA7 causes increased numbers of adult neural stem cells, it was suggested ( Holmberg et al., 2005 ) that ephrin-A reverse signaling normally negatively regulates adult neural stem cell proliferation. It is not yet clear whether ephrin-B reverse signaling functions to promote or block the maintenance of adult neural stem cells. Future analyses on single or double knockout mice of ephrin-B1 and -B2 (ephrin-B1 and -B2 are both expressed in the SVZ neural stem cells; Conover et al., 2000 ;Ricard et al., 2006 ) will help to better understand this.

Mechanism of the ephrin-B/RGS function in cortical neural progenitor cells
During cortical neurogenesis, neural progenitor cells in the VZ go through consecutive rounds of cell cycles over an extended period of time. Detailed cell cycle measurements reveal that (percentage of short pulse-labeled BrdU + S-phase cells per 40 × optical view; Chenn and Walsh, 2002 ) and the mitotic index (percentage of p-H3 + mitotic cells per 40 × optical view; Li et al., 2003 ) to look at the effects of ephrin-B1 knockout in neural progenitor cell population. We found that mutant (null or het) brains showed a lower BrdU labeling index ( Fig. 6 B ) and a decrease in the mitotic index ( Fig. 6 C ) compared with the wild-type brains, indicating that there were fewer neural progenitor cells in the ephrin-B1 mutant brains. Tunnel staining showed that there was no obvious difference in cell apoptosis between mutant and wild-type brains (unpublished data). To further address the possible mechanisms of neural progenitor cell depletion in the ephrin-B1 mutant mice, we performed a 24-h labeling of BrdU to examine cell cycle exit and reentry by measuring the fraction of BrdU + Ki67 Ϫ cells (cells that have exited cell cycle) in BrdU + cells per 40 × optical view ( Chenn and Walsh, 2002 ). As shown in Fig. 6 D , the mutant brains revealed a marked increase in the fraction of progenitor cells exiting the cell cycle (more BrdU + Ki67 Ϫ cells), thus reducing the pool of self-renewing progenitor cells. These results indicate that genetic loss of function of ephrin-B1 causes progenitor cells to leave the cell cycle and leads to a loss of cortical neural progenitor cells in development.

Discussion
Using cellular, embryological, and genetic approaches, we have observed that gain of function of ephrin-B/PDZ-RGS3 signaling prevents differentiation of cortical neural progenitor cells, whereas loss of function induces differentiation. Our fi ndings thus identify ephrin-B1 as a functional surface marker for neural progenitor cells in the developing cerebral cortex and indicate that ephrin-B/PDZ-RGS3 signaling plays a critical role in the maintenance of the neural progenitor cell state during brain development.

Ephrin-B reverse signaling in the cerebral cortex
Our study has uncovered a previously unknown function of ephrin-B in development: maintaining the pool of cortical neural progenitor cells. This role of ephrin-B was fi rst suggested by the specifi c expression pattern of ephrin-B1 and its downstream signaling mediator PDZ-RGS3 in cortical neural progenitor cells. Cellular and embryological studies next showed that ephrin-B/ PDZ-RGS3 signaling can suffi ciently block differentiation and is important for maintaining neural progenitor cell identity. Consistent with these observations, knockout of ephrin-B1 causes a reduction in the number of neural progenitor cells in the embryonic cortices.
The ability of ephrin-B to initiate signaling in both directions (to activate a forward signaling via Eph and initiate a reverse signaling through its own cytoplasmic domain; Flanagan and Vanderhaeghen, 1998 ;Drescher, 2002 ;Poliakov et al., 2004 ;Davy and Soriano, 2005 ;Egea and Klein, 2007 ;Arvanitis and Davy, 2008 ;Pasquale, 2008 ) implies that an ephrin-B mutant phenotype could be caused by a cell-autonomous or -nonautonomous function. For example, the functions of ephrin-B in hindbrain segmentation ( Xu et al., 1999 ), in the formation of the One study suggested that the G ␤ ␥ subunits control the mitotic spindle orientation in cortical neural progenitor cells thereby inhibiting differentiation ( Sanada and Tsai, 2005 ). In Caenorhabditis elegans and Drosophila melanogaster , increasing genetic evidence has implicated a fundamental role for G proteins in controlling neural progenitor asymmetrical cell division ( Wilkie and Kinch, 2005 ;Yu et al., 2006 ), which governs selfrenewal and differentiation. In this function, G ␣ subunit, Pins/ AGS3/LGN, GoLoco/GPR, Ric-8, and RGS proteins work together in a noncanonical G protein pathway to control asymmetrical division in progenitor differentiation ( Wilkie and Kinch, 2005 ;Yu et al., 2006 ). In view of the G protein pathway from D. melanogaster and C. elegans , our results suggest that a similar mechanism of G ␣ subunits and RGS proteins may function in the mammalian neural progenitor cells, including likely candidate participants PDZ-RGS3 and G ␣ i2 subunit, which was shown to be selectively expressed in the VZ of the developing cerebral cortex ( Asano et al., 2001 ;unpublished data). Our results would further suggest that the RGS function in the noncanonical G protein pathway may be modulated by an upstream receptor molecule, such as ephrin-B. This was refl ected in our gain-of-function study that both ephrin-B and PDZ-RGS3 are required for maintaining the neural progenitor state.

Selection of shRNAs
Candidate shRNAs of ephrin-B1 were cotransfected into HEK cells with psi-CHECK (Promega) -ephrin-B1. Cell extracts were made 48 h after transfection and the potency of shRNAs was assessed using dual luciferase activities. Inhibition on ephrin-B1 expression was confi rmed using Western blot on transfected cells (EGFP was coexpressed with shRNAs in pNUTs and was used as a loading control) or immunocytochemistry on cortical progenitor cells. The sequences of shRNAs used in the functional assays were the following (the loop of shRNA and the mutations introduced are underlined and bolded, respectively): shRNAB1.3, ACCAUCAAGUUCC A A G A-G U UU C A A G A G A ACUCUUGGAACUUGAUGGU; shRNAB1.3mt, A C C A-U C A A U C C CC A A G AGU UUCAAGAGA ACUCUUG G G GA UUG AU GGU; shRNAB1-3UTR, G A CUGCUCGUCCACUAUCA UUCAAGAGA UGAUA G-U G G A C G A G C A G UC; and shRNAB1-3UTRmt, GA UCAU UCGUCCACU-AUCA UUCAAGAGA UGAUAGUGGACGA AUGA UC.

RNA in situ and immunohistochemistry
RNA probes for ephrin-B1, PDZ-RGS3, and EphBs were described in Lu et al. (2001) . RNA in situ and immunohistochemistry were done essentially as in early in neurogenesis, neurons are generated relatively slowly and the VZ expands rapidly through accumulation of progenitor cells but the reverse happens at a later stage ( Takahashi et al., 1996 ). A general model of cortical neurogenesis ( McConnell, 1995 ;Takahashi et al., 1996 ;Monuki and Walsh, 2001 ;Temple, 2001 ;Gupta et al., 2002 ;Fishell and Kriegstein, 2003 ;Gotz and Huttner, 2005 ;Rakic, 2006 ;Molyneaux et al., 2007 ) suggests that in the early phase, neural progenitor cells predominantly go through symmetrical divisions to generate more progenitor cells. Then cell divisions progressively shift to asymmetrical generation of one progenitor and one neuron. In the fi nal phase of neurogenesis, progenitor cells might also divide symmetrically to give rise to two daughter neurons to expand the neuronal population more quickly. Our results suggest that ephrin-B/PDZ-RGS3 signaling is likely to have a regulatory role at the stage when neural progenitor cells are shifting from progenitor selfrenewal by symmetrical cell division to generation of neurons by asymmetrical cell division.
The dominant-negative inhibition by a mutant PDZ-RGS3 protein that lacks the RGS domain ( Fig. 5 ) indicates that the function of ephrin-B/PDZ-RGS3 signaling in neural progenitor cells is likely mediated through inhibition of the heterotrimeric G protein signaling ( De Vries et al., 2000 ;Ross and Wilkie, 2000 ;Siderovski and Willard, 2005 ). Our results thus suggest the following working model: the RGS-mediated ephrin-B signaling is required to promote neural progenitor maintenance via inhibition of G protein signaling ( Fig. 7 ). This model proposes that the G ␣ subunit of the heterotrimeric G proteins functions as an activator of neuronal differentiation during cortical neurogenesis, perhaps through promoting asymmetrical cell division. It is interesting to note that ephrin-Eph signaling was recently shown to mediate the establishment of asymmetrical cell fates in the Ciona intestinalis notochord and neural tube ( Picco et al., 2007 ;Shi and Levine, 2008 ).
The involvement of heterotrimeric G proteins in the mammalian neural progenitor cell fate determination is not clear. Our results indicate that the RGS-mediated ephrin-B reverse signaling pathway is required for the maintenance of neural progenitor cells. As the RGS domain acts as an inhibitor of G ␣ subunit signaling, our data suggest that G ␣ subunit may function as an activator of progenitor differentiation. Future studies are required to investigate this potential role of G ␣ subunit. In addition, whether and how EphB functions in the activation of ephrin-B -RGS pathway in neural progenitor cells will also need to be further studied.

Image acquisition and processing
Immunohistochemistry was performed on frozen sections using Cy2 or Rhodmine Red-X -conjugated secondary antibodies. Slides were mounted in aqueous mounting media. Fluorescent images were all acquired using the confocal microscope (LSM 510 Upright 2 photon) with 0.5 NA FLUAR10 × , 0.8 NA Plan-APOCHROMAT 20 × , 0.75 NA Plan-NEOFLUAR 40 × , 1.4 NA Plan-APOCHROMAT (digital image contrast) 63 × oil immersion, and 1.4 NA Plan-APOCHROMAT 100 × oil immersion objectives. Digital images were processed (brightness/contrast adjusted and color balanced) using Image-Pro Plus 5.1 software (Media Cybernetics, Inc.). RNA in situ images were captured on a dissection scope (S6D; Leica) connected to a camera (Spot Insight; GE Healthcare). Spot basic Version 3.5.5 was used for RNA In situ image processing.

In utero electroporation functional analysis
In utero electroporation was performed on E13.5 embryos as described previously ( Ohtsuka et al., 2001 ;Saito and Nakatsuji, 2001 ). Electroporated brains were dissected out at E14.5 or at a later stage, cryoprotected, sectioned, and stained with HOECHST for microscopic analyses. For BrdU pulse labeling, 50 mg/kg BrdU was intraperitoneally injected into the female mouse 1.5 h before brain dissection. For cell cycle exit analysis, BrdU was administered at E14.5 and the electroporated brains were dissected out for analysis at E15.5. Electroporated cells were detected by direct visualization of EGFP expressed from the shRNA vector or by immuno histochemistry on EGFP that was expressed as a fusion protein to ephrin-B1 or PDZ-RGS3. Quantifi cation of the distribution of green cells in the cortex was done using Image-Pro Plus 5.1.

Neural progenitor cell culture, transfection, and immunostaining
Cerebral cortices of E11.5 mouse embryos were dissected out in HBSS. Cells were dissociated by trituration using a pipette with a blue tip. After centrifugation at 1,000 rpm for 5 min, cells were washed twice with HBSS. Cell pellets were then resuspended in DME/F12 medium supplemented with 1:50 vol/vol B27, 100 units/ml penicillin, and 100 μ g/ml streptomycin, counted, plated (2 × 10 5 cells/well) onto 15 μ g/ml polyornithine in H 2 O and 2 μ g/ml fi bronectin (in PBS)-coated coverslips placed in a 24well plate, and cultured at 37 ° C. 1 d after plating, cells were transfected in triplicate with shRNAB1.3 or shRNAB1.3mt using Fugene-6 (Roche). 2 d after transfection, cells were fi xed with 4% PFA for immunocytochemistry. For BrdU labeling, BrdU was added to the culture at a fi nal concentration of 10 μ M, 16 h (overnight) before fi xation. The acutely dissociated cells were prepared as described in a previous study ( Hartfuss et al., 2001 ).

Transwell analysis
EGFP or EGFP-PDZ-RGS3-IRES-HA-ephrin-B1 electroporated brains were removed at E15.5. The cortical areas that express the electroporated plasmid were dissected out under a fl uorescent microscope and dissociated in HBSS. After being washed twice, cells were resuspended in DME/F12 medium supplemented with B27. Transwell migration assay was performed essentially as described previously .

Mutant ephrin-B1 mice analysis
Ephrin-B1 knockout mice were reported previously ( Davy et al., 2004 ). BrdU labeling index, mitotic index, and progenitor cell cycle exit index were analyzed essentially as described previously ( Chenn and Walsh, 2002 ;Li et al., 2003 ). In brief, to obtain BrdU labeling index and progenitor cell cycle exit index, pregnant female mice were labeled with 50 mg/kg BrdU for 2 and 24 h, respectively. Cryosections of the brains (12 -14 μ m) were processed for BrdU and Ki67 staining and images were taken using a confocal microscope (LSM 510 Upright 2 photon). For BrdU labeling index, BrdU + cells were counted against the total cells stained by propidium iodide in a 40 × optical view of each section. For progenitor cell cycle exit index, BrdU + Ki67 Ϫ cells (cells exiting cell cycle) were counted against the total BrdU + cells/40 × optical view. For mitotic index, p-H3 + cells were counted against the total cells stained by HOECHST in 40 × optical views. Fig. S1 shows electroporation-mediated gain of function of ephrin-B1 and PDZ-RGS3 in the cortex using EF promoter -based vector. Fig. S2 shows specifi c inhibition of ephrin-B1 expression by two different shRNAs. Online supplemental material is available at http://www.jcb.org/cgi/ content/full/jcb.200708091/DC1.


extfieldsofstudy: Biology Medicine

s2fieldsofstudy: Biology Medicine

text:
Assessment of decorin-binding protein A to the infectivity of Borrelia burgdorferi in the murine models of needle and tick infection

Background Decorin-binding proteins (Dbps) A and B of Borrelia burgdorferi, the agent of Lyme disease, are surface-exposed lipoproteins that presumably bind to the extracellular matrix proteoglycan, decorin. B. burgdorferi infects various tissues including the bladder, heart, joints, skin and the central nervous system, and the ability of B. burgdorferi to bind decorin has been hypothesized to be important for this disseminatory pathogenic strategy. Results To determine the role of DbpBA in the infectious lifecycle of B. burgdorferi, we created a DbpBA-deficient mutant of B. burgdorferi strain 297 and compared the infectious phenotype of the mutant to the wild-type strain in the experimental murine model of Lyme borreliosis. The mutant strain exhibited a 4-log decrease in infectivity, relative to the wild-type strain, when needle inoculated into mice. Upon complementation of the DbpBA-mutant strain with DbpA, the wild-type level of infectivity was restored. In addition, we demonstrated that the DbpBA-deficient mutant was able to colonize Ixodes scapularis larval ticks after feeding on infected mice and persist within the ticks during the molt to the nymphal state. Moreover, surprisingly, the DbpBA-mutant strain was capable of being transmitted to naïve mice via tick bite, giving rise to infected mice. Conclusion These results suggest that DbpBA is not required for the natural tick-transmission process to mammals, despite inferences from needle-inoculation experiments implying a requirement for DbpBA during mammalian infection. The combined findings also send a cautionary note regarding how results from needle-inoculation experiments with mice should be interpreted.

Background
The causative agent of Lyme disease, Borrelia burgdorferi, is introduced into a mammalian host via tick bite, whereupon the organisms enter the skin, disseminate hematogenously, and persist in the presence of a strong host immune response [1]. The dissemination and persistence of B. burgdorferi within a mammalian host is thought to be predicated, at least in part, on the organism's ability to bind molecules of the extracellular matrix (ECM) [2][3][4], inasmuch as these interactions have been shown to be important for other bacterial pathogens [5]. Among various ECM components, B. burgdorferi binds to type I collagen [4], fibronectin [6,7], integrins [8,9], the proteoglycan decorin [10], and glycosaminoglycans (GAGs) [11,12]. The B. burgdorferi proteins described as ECM-binding proteins include BBK32 [6,13], Bgp (borrelia-GAG binding protein) [12], P66 (Oms66) [14], decorin-binding protein (Dbp) A, and DbpB [10,15]. These proteins, and perhaps other as yet unidentified molecules, may play a significant role in the infectivity and pathogenesis phenotypes of B. burgdorferi.
Our laboratory has been interested in DbpA as a vaccine candidate for the prevention of Lyme disease and the contribution of DbpA and DbpB to B. burgdorferi pathogenesis and infectivity [16,17]. Since their in vitro characterization as decorin-binding proteins [10,15], the DbpA and DbpB lipoproteins have been implicated as potential contributors to adhesion and colonization of B. burgdorferi within mammalian hosts [15,18]. The genes that encode DbpA and DbpB reside in an operon, dbpBA, on linear plasmid 54 (lp54) and are found within many B. burgdorferi sensu lato isolates [19]. Neither protein is expressed by B. burgdorferi within the tick vector [17], however, expression of dbpA (and presumably dbpB) is upregulated in the mammalian host after ticks deposit spirochetes into the skin [20,21]. DbpA and DbpB expression likely remains high for the duration of mammalian infection, as inferred by the presence of antibodies against both antigens in the serum of mice as late as one year after infection (Hagman, unpublished data). The presence of antibodies against DbpA in the serum of patients with late-stage, disseminated Lyme disease also is well-documented [22], providing added support for DbpA expression by B. burgdorferi during chronic infection.
Although the combined data to date suggest an important role for the decorin-binding proteins of B. burgdorferi during mammalian infectivity, virtually all data inferring the importance of DbpA and DbpB thus far have been indirect. The first direct investigation into the role of the dbpBA operon in the infectious lifecycle of B. burgdorferi was carried out by Shi et al [23]. In this study, mutational analysis of dbpBA in B. burgdorferi strain B31 indicated that neither DbpA, nor DbpB was essential in the murine needle-challenge infection model of borreliosis. However, there was evidence suggesting that these mutants exhibited a modest level of attenuation in immunocompetent mice. Unfortunately, a comprehensive 50% infectious dose (ID 50 ) was not included in this report to further investigate this possible defect, nor was genetic complementation of the mutation performed. This latter point is of particular importance given the genetic plasticity of B. burgdorferi and the possibility that spontaneous loss of an endogenous borrelial plasmid might account for the apparent defect in this mutant. To more directly examine the role of both DbpA and DbpB in the murine/tick model of Lyme disease, a dbpBA-deficient mutant and a dbpA genetic complement of the mutant were generated in the infectious strain 297 of B. burgdorferi. Phenotypic assessment of mouse needle infectivity by ID 50 analysis and Ixodes scapularis tick colonization and tick-transmis-sion capacity by the Bb297 dbpBA-mutant also were performed.

Construction and characterization of a dbpBA-deficient mutant
To assess the roles that DbpA or DbpB play in the infectivity and pathogenesis of B. burgdorferi, we created a dbpBAdeficient mutant by allelic exchange of a PflgB-kan cassette for the majority of the dbpBA operon (Fig. 1A). Infectious Bb297 was chosen for construction of the dbpBA mutant because it is a human isolate [24] and prior infectious mutants of this strain have been readily created [25][26][27][28].
Bb297 was electroporated with the suicide vector, pKHd-bpAko, containing a PflgB-kan cassette flanked by 1.5 kb of DNA on the left side of the dbpBA operon and 890 bp of DNA on the right side of the dbpBA operon (Fig. 1A). This allelic exchange strategy relies on a double crossover, homologous recombination event to replace the dbpBA operon with the PflgB-kan cassette. Several kanamycinresistant transformants were obtained, and the presence (Fig. 1B) and orientation (Fig. 1C) of the PflgB-kan cassette within the dbpBA operon was confirmed by PCR analysis.
During genetic manipulation and in vitro cultivation, B. burgdorferi may spontaneously lose endogenous plasmids that are not required for growth in vitro, but are essential for mammalian infection. At least two plasmid-encoded genes, vlsE [29] and pncA (BBE22) [30], fit this criteria. One kanamycin-resistant transformant, BbKH500, retained both vlsE and pncA (Fig. 2) and was analyzed by immunoblotting to confirm the loss of DbpA and DbpB. As expected, DbpA and DbpB were absent from BbKH500 (Figs. 3A and 3B). BbKH500 exhibited identical doubling times when compared to wild-type Bb297 (data not shown) and PCR-based plasmid profiling revealed that the endogenous plasmid profile of BbKH500 matched that of the parent Bb297 (Fig. 2).

Loss of DbpBA significantly reduces the infectivity of B. burgdorferi in mice when introduced via needle inoculation
To determine the contribution of DbpA and DbpB to the infectivity of B. burgdorferi in mice, C3H/HeJ mice were challenged intradermally via needle inoculation with increasing numbers of BbKH500 (10 4 , 10 5 , 10 6 and 10 7 spirochetes). As a positive control, an additional five mice were inoculated with wild-type Bb297 at a dose of 10 3 spirochetes (ID 50 of approximately 50 spirochetes; [27]). The results are shown in Table 1. Whereas the mice infected with 10 3 of Bb297 showed culture positive ear punch biopsies at two weeks post-infection, needle challenge of naïve mice with 10 4 BbKH500 did not produce an infec-Construction of dbpBA-deletion mutant BbKH500 and Prom-dbpA complementation vector and PCR confirmation Figure 1 Construction of dbpBA-deletion mutant BbKH500 and Prom-dbpA complementation vector and PCR confirmation. (A) Strategy for the replacement of the dbpBA operon with the PflgB-kan and complementation with pKH2000. pKH-dbpBAko was the pGEM-T easy-based suicide plasmid used to transform Bb297 for the homologous recombination of the kanamycin-resistance gene into the dbpBA operon. "A" denotes AscI sites. PflgB-kan denotes the kanamycin-resistance marker expressed from the flgB promoter. The borrelial shuttle vector containing the dbpBA Prom fused to the dbpA ORF (pKH2000) was transformed into BbKH500 to restore DbpA expression. Oligonucleotide primers used for PCR are indicated with short arrows. (B) PCR using primers ko5 and ko4 (shown in panel A). The first two lanes are undigested PCR products from Bb297 and BbKH500, whereas the second two lanes are the corresponding PCR products digested with AscI. (C) Lanes 1, 3 and 5 are PCR products derived from BbKH500 template DNA and lanes 2, 4 and 6 are PCR products derived from Bb297 template DNA. Primer pairs used in PCR are indicated above the lanes.  dbpA PflgB-kan dbpA tion in any mouse (n = 15), even when the infection was allowed to progress for 14 weeks. Seventeen mice challenged with 10 5 and ten mice challenged with 10 6 BbKH500 also did not show signs of infection (negative ear-punch biopsy culture) at four weeks post-challenge (Table 1). However, after 10 weeks, three mice from the group infected with 10 5 BbKH500 and two mice from the group challenged with 10 6 BbKH500 were shown to be infected by positive ear-punch biopsy cultures. Additionally, mice were challenged with either 10 5 or 10 6 BbKH500 and ear-punch biopsies were harvested 14 weeks after infection (Table 1). From the group that received 10 5 spirochetes, two mice were infected (n = 10), and from the group that received 10 6 spirochetes, four mice were infected (n = 10). All of the mice challenged with 10 7 of BbKH500 had positive ear-punch cultures as early as five-weeks post-challenge, and as late as 14 weeks post-infection (n = 5). These spirochetes from ear punchpositive cultures were analyzed by diagnostic PCR as described above, and confirmed to be Bb297 or BbKH500 (data not shown). Aliquots of these cultures also were passed to media containing kanamycin to confirm resistance and sensitivity of BbKH500 and Bb297, respectively. Seroconversion analyses performed on a subset of mice also revealed that none of the mice infected with BbKH500 showed antibody reactivity against DbpA or DbpB (data not shown). Furthermore, only mice that exhibited culture-positive ear punch cultures showed significant serum antibody reactivity with P39 (data not shown). Analysis of all infection results yielded an ID 50 for BbKH500 of 1.2 × 10 6 (p <0.001) compared with ID 50 of approximately 50 bacteria for wild-type Bb297 [27].

Complementation of the dbpBA-deletion mutant with DbpA restores infectivity in needle-challenged mice
The results above suggested that the dbpBA operon is required for full infectivity of B. burgdorferi when mice are infected via needle inoculation. However, during genetic manipulation of B. burgdorferi, it is not uncommon to lose one or more plasmid(s) which potentially contribute to infection, therefore genetic complementation is necessary to definitely ascribe the attenuated phenotype in BbKH500 to the dbpBA lesion. Although BbKH500 carries a mutation in both DbpA and DbpB, we chose to complement with only dbpA because i) DbpA is better characterized that DbpB [31,32] and ii) experimentation suggests that DbpA is the prominent Dbp in B. burgdorferi [15,33]. To restore DbpA expression in BbKH500, a shuttle plas-PCR-based plasmid profiling to compare the plasmid contents of the strains employed in this study Figure 2 PCR-based plasmid profiling to compare the plasmid contents of the strains employed in this study. PCR amplification with primers specific for each of the known endogenous B. burgdorferi plasmids was used to compare the plasmid content of parent Bb297, BbKH500, and BbKH501. Sequence information for the primers utilized is provided in Table 4 mid carrying a copy of dbpA driven by its native promoter was constructed (Fig. 1A). Since dbpA is the second gene in the dbpBA operon, we chose to clone the promoter for this operon upstream of the dbpA gene and then insert the fusion product in the multiple-cloning site of pJD51. BbKH500 cells were transformed with this shuttle plasmid (pKH2000), yielding clones resistance to both kanamycin and streptomycin. These transformants were screened by PCR to verify the presence of the complementing plasmid (Fig. 3C). Clones shown to contain the complementing plasmid were checked for vlsE and pncA (Fig 2.), as described above. PCR-based plasmid profiling, performed on one of the transformants that contained both pncA and vlsE, revealed a profile identical to the parent strain, BbKH500 (Fig. 2). This clone, designated BbKH501, was selected for further analysis and DbpA expression was confirmed by both SDS-PAGE ( Fig. 3A) and immunoblot analysis (Fig. 3B). Proteinase K digestion of BbKH501 and Bb297, under conditions that left FlaB intact, demonstrated surface exposure of DbpA in both strains (Fig. 3D). Three groups of five C3H/HeJ mice were needle inoculated (i.d.) with BbKH501 at 10 2 , 10 3 or 10 4 borreliae per mouse. Ear punch biopsies were harvested at either two weeks (10 3 and 10 4 doses) or three weeks (10 2 dose); all 15 mice became infected, as confirmed by positive ear-punch biopsy cultures. Although a precise determination of the ID 50 of BbKH501 could not be calculated, the fact the 100% of the mice were infected with 10 2 bacteria suggests that the ID 50 is less than 10 2 spirochetes (4-logs lower than BbKH500). Aliquots of these cultures were passed to media containing streptomycin and kanamycin selection to confirm resistance. Diagnostic PCR also confirmed that the spirochetes growing out of the ear punch biopsy were BbKH501 (data not shown). Seroconversion analyses carried out on a subset of mice also revealed that the mice infected with BbKH501 showed antibody reactivity against DbpA, but not DbpB (data not shown). These data confirm that DbpA is necessary for wild-type levels of B. burgdorferi infectivity in mice when introduced via needle inoculation, and that DbpB is dispensable for this aspect of the infectious process.

Neither DbpA nor DbpB is required for acquisition of B. burgdorferi by ticks or infection of mice via tick bite
To determine whether I. scapularis ticks could acquire borreliae lacking dbpA and dbpB, naïve larvae were allowed to feed to repletion on mice infected with BbKH500 or on mice infected with Bb297. Larvae were collected after feeding, allowed to molt, and the unfed nymphs were examined for the presence of B. burgdorferi by IFA; approximately 11 weeks had elapsed since these ticks had fed to repletion as larvae. Microscopic examination of multiple fields revealed approximately one spirochete per field in the BbKH500-infected ticks (23 ticks examined), as opposed to 20-30 spirochetes per field in the Bb297infected ticks (10 ticks examined). All Bb297-infected ticks examined were positive for B. burgdorferi. Of the ticks that had fed on BbKH500-infected mice, 80% were positive for spirochetes. Characterization of BbKH500 and BbKH501 Because the ticks maintained strain BbKH500 through their molt, it was important to assess whether the loss of DbpA and DbpB affected B. burgdorferi transmission to mice via tick bite. To test this, five BbKH500-infected nymphs were placed on each of two naïve mice and ten BbKH500-infected nymphs were placed on each of six naïve mice. An additional two naïve mice received five Bb297-infected ticks each. The ticks were allowed to feed to repletion, and at three weeks post-infestation, earpunch biopsies were harvested from all mice. The earpunch biopsies were cultured in BSK medium and the cultures examined by dark-field microscopy for the presence of B. burgdorferi. These results are shown in Table 2. One of the two mice infested with five ticks harboring BbKH500 was infected as demonstrated by a positive earpunch culture, and four of the six mice infested with ten ticks harboring BbKH500 were infected. Both of the mice on which the Bb297-infected ticks fed had positive earpunch biopsy cultures. These spirochetes were analyzed by diagnostic PCR as described above, and confirmed to be Bb297 or BbKH500 (data not shown). Seroconversion analyses performed on a subset of the BbKH500-infected mice also revealed that none of the mice infected with BbKH500 showed antibody reactivity against DbpA or DbpB (data not shown). Furthermore, only mice that exhibited culture-positive ear punch cultures showed significant antibody reactivity against P39 (data not shown). Taken together, these data indicate that DbpA and DbpB are not essential for the transmission of B. burgdorferi from ticks to mice.

Discussion
The importance of ECM-binding proteins to the pathogenic strategy of B. burgdorferi is not currently known. It is presumed that borreliae bind mammalian host cells during infection because binding to various ECM molecules and to tissue culture cells has been demonstrated in vitro [4,34,35]. Of the known ECM-binding proteins of B. burgdorferi, Bgp [36] and BBK32 [37] have been deleted from infectious strains N40 and B31, respectively, and shown to be dispensable for infectivity in the mouse model of Lyme disease. A deletion of integrin-binding protein, P66, in a noninfectious strain of B. burgdorferi, HB19 [38], caused the spirochetes to lose their ability to bind b 3chain integrins [14] but the phenotype of a P66 mutant in an infectious strain of B. burgdorferi is currently unknown. The remaining two known ECM-binding proteins, DbpA and DbpB, were the focus of this work due to multiple lines of evidence suggesting a role for one or both proteins during infection of mammalian hosts [15,18,31]. To directly address the contribution of both DbpA and DbpB to B. burgdorferi infectivity, we created BbKH500, a dbpBAdeletion mutant of the human isolate Bb297, and used this strain to challenge mice with increasing numbers of spirochetes. We observed a 4-log reduction in infectivity of the mutant strain when the spirochetes were needle inoculated into mice. Complementation of the dbpBAmutant with DbpA alone in the dbpBA-mutant restored infectivity of B. burgdorferi to wild-type levels.
Prior to this work, Fischer et al. [18] had restored DbpA and DbpB expression (via shuttle plasmid) to B. burgdorferi strain B314, a non-adherent, noninfectious derivative of strain B31 that has lost several plasmids, including lp54 [39]. Although expression of the Dbp molecules restored binding of the spirochetes to purified decorin, dermatan sulphate, and human epithelial cells [18], experiments to test the infectivity of B314 expressing DbpA and DbpB in the murine model of Lyme disease were implausible due to the loss of plasmids that are required for mammalian infection [39]. A recent study by Shi et al. [23] assessed the infectivity of a DbpA,B mutant of B. burgdorferi strain 5A18NP1, a B31 derivative that lacks the BBE02 gene [40]. Based on needle-challenge results obtained from a single inoculation dose of 10 5 bacteria (80% infection), Shi et al. [23] ascertained that the dbpBA locus was not required for infectivity. However, in the present study, we observed a 4-log increase in the ID 50 of BbKH500 (>10 6 bacteria) and a 19% infection rate with a dose of 10 5 BbHK500 when mice were infected by needle inoculation. The reason for the difference in infectivity levels of the dbpBA-deficient mutants reported by Shi et al. and BbKH500 created in our laboratory is unknown at this time. One possible explanation for this disparity could be due to our use of Bb297 as the parental strain for the present mutational analysis, whereas Shi et al. [23] utilized a clonal derivative of B31 that is a BBE02-mutant. This variability also could be explained by spirochete enumeration differences prior to needle challenge. As mentioned in the Methods, significant attention was given to the enumeration of BbKH500 prior to needle inoculation of mice.
It should be noted that during the course of the study described herein, a subsequent report on the role of DbpBA was published by Shi et al. [41], which, unlike their previous study, included genetic complementation experiments. Our current findings agree with those of this most recent report in that Shi et al. also observed a 4-log a Varying number of ticks infected with BbKH500 were allowed to feed to repletion on naïve mice. b Infection rates were assessed at three weeks post-infestation by culturing BbKH500 from ear-punch biopsies increase in ID 50 values with their dbpBA mutant [41]. In addition, Shi et al. observed a defect in the ability of the dbpBA mutant to colonize heart, joint and skin tissues, suggesting an overall deficiency in dissemination [41]. The observation that mice infected with BbKH500 showed a delay in infectivity (10-to 14-weeks post-infection) also suggests that BbKH500 might be attenuated with respect to its capacity to disseminate through the host. However, whereas we were able to compensate fully for the loss infectivity in the dbpBA mutant by complementing with dbpA alone (as assessed by ear punch biopsy culture), Shi et al. reported that both dbpA and dbpB were required to restore the infectivity of their dbpBA mutant [41]. Although the precise reason for this disparity is unknown at this time, there are several differences between the experimental approaches of these studies that might account for these differing results. First, it is possible that some intrinsic differences(s) in the bacterial strains utilized might account for the variation observed between these two studies. Specifically, as noted above, strain 297 was the parental strain used in the current study, and Shi et al. utilized a highly-transformable clone (5A13) of strain B31 that lacks both lp56 and the virulence-associated plasmid lp25 as the background for their mutagenesis experiments [30,41]. Second, the disparity in our results might be due to the use of different strains of mice in these two studies; C3H/HeJ mice were used in our study, whereas Shi et al used BALB/c [41]. This may be relevant because numerous studies have reported that experimental infection of C3H strains of mice with B. burgdorferi results in significantly different disease pathologies [42,43], higher spirochetal loads in multiple tissues [44], and different cytokine responses [45,46] by comparison to similarly infected BALB/c mice. Although, it is difficult to precisely predict how the reported differences in pathogenesis observed in these two distinct murine backgrounds might be impacted by DbpA and/or DbpB during infection, it is reasonable to suspect that these differences might account for some of the disparity in the results of complementation experiments obtained in the aforementioned studies.
Even though DbpA was required for full infectivity by Bb297 in mice when introduced by needle inoculation (intradermally), neither DbpA, nor DbpB, was required for infection of larval I. scapularis ticks or for transmission of BbKH500 from infected ticks to naïve mice. That DbpA was not required for uptake of the spirochetes by larval ticks was not surprising due to our earlier work demonstrating that DbpA is not expressed by B. burgdorferi harbored within the tick vector [17]. However, based on the data we obtained from needle inoculation of mice with BbKH500, it was surprising that as few as five nymphal ticks could transmit BbKH500. This is especially surprising because semi-quantitative analysis of the total number of spirochetes observed in the midguts of unfed ticks infected with BbKH500 showed a lower spirochete density (approx. 1/field) by comparison to number of bacteria present in the dissected midguts of the Bb297-infected ticks (20-30/field). While these data might suggest a possible defect in either the efficiency of acquisition of the dbpBA mutant or the capacity of this mutant to persist in the tick midgut, there were still sufficient numbers of the mutant spirochetes in these ticks to infect naïve mice.
Since it is not known precisely how many spirochetes are transmitted by ticks during the feeding process [47], it was impossible to directly compare the number of B. burgdorferi transmitted by tick bite to our needle-challenge experiments. Spirochete numbers within salivary glands during tick feeding have been estimated between 20 [47] and 61 spirochetes [48] per salivary gland pair. These data suggest that an individual tick deposits far fewer than 10 4 spirochetes during the feeding process, and our results have shown that by the needle-inoculation route, 10 4 BbKH500 are not infectious.
In addition to the data presented by Fischer et al. [18], additional reports have attempted to address the role of DbpA, DbpB, and the ECM proteoglycan, decorin, with respect to the infectivity and pathogenesis of B. burgdorferi. Brown et al. described B. burgdorferi infection of decorin-deficient mice (Dcn -/-) [49]. Brown et al. [49] found that, by comparison to Dcn +/+ mice, Dcn -/mice challenged with a higher dose of B. burgdorferi (10 4 ) had i) fewer infected joints, ii) a reduction in the severity of arthritis, but iii) no significant defect in colonization of the other tissues. Whereas 10 4 wild-type B. burgdorferi were infectious for Dcn -/mice in the studies by Brown et al. [49], the Bb297 dbpBA-mutant was unable to infect mice at this dose. When considered together, these data support the hypothesis that DbpA, in addition to binding decorin, may have an additional ligand(s) or has another function critical for infectivity. At the present time, the only other known ligand recognized by DbpA is the GAG dermatan sulfate [18], but the contribution of this interaction to the pathogenesis of B. burgdorferi remains to be elucidated.

Conclusion
Despite the disparities between the results of the complementation experiments described in the current study and those obtained by Shi et al. [41], the overall results of our needle-inoculation experiments are in agreement with the most recent conclusion of Shi et al. that mutation of dbpBA results in significant attenuation of B. burgdorferi in the murine model of Lyme borreliosis. However, the observation that the dbpBA mutant showed a significant reduction in infectivity when the mice were needle inoculated is overshadowed by the finding that this same mutant was capable of infecting mice via tick challenge.
The fact that DbpA and DbpB are dispensable for infection via the tick-mediated route of infection suggests that B. burgdorferi transmitted via tick bite are in some way phenotypically different than their in vitro-cultivated counterparts, and/or that tick-derived salivary components, such as Salp15, may assist B. burgdorferi during the early infection process [50]. Taken together, these results emphasize the importance of characterizing the impact of a given gene in the infectious lifecycle of B. burgdorferi using the natural tick vector, as opposed to using only the artificial needle-challenge model.

Generation of DbpBA deletion mutant in Bb297
The dbpBA-deficient Bb297 strain was created by allelic exchange of the dbpBA operon with a kanamycin-resistance cassette, PflgB-kan [53], derived from pBSV2 [54]. The mutagenesis construct, pKHdbpBAko, was created by generating two PCR products that constituted the left and right flanking regions of the dbpBA operon which then were joined via an AscI restriction site, thus deleting a significant portion of the dbpBA operon. Takara EX Taq polymerase (Takara Bio Inc., Shiga, Japan) and oligonucleotide primers ko1 and ko2 (Table 3) were used to amplify the left arm and ko3 and ko4 (Table 3) were used to amplify the right arm; primers ko2 and ko3 were modified such that AscI restriction sites would be introduced into the "middle termini" of the two PCR fragments. The resulting PCR products then were digested with AscI and ligated together. The linear ligated product was used as the template in a second PCR amplification containing the primers ko1 and ko4. The resulting PCR product, representing the joined flanking regions, was cloned into pGEM-T Easy vector (Promega Corp., Madison, WI) to generate pGEMT-dbpBA-AscI. The PflgB-kan cassette was from pJD55, a derivative of pJD44 in which the original aph [3']-IIIa was replaced with the PflgB-kan cassette of pBSV2 [27,54]. In pJD55, the PflgB-kan cassette is flanked by AscI sites which facilitated the cloning of the marker into the unique AscI site within pGEMT-dbpBA-AscI to create pKHdbpBAko. Primers kan5' and kan3' were used with primers ko1 and ko4 to determine the orientation of the PflgB-kan cassette with respect to the dbpBA operon.
Bb297 were made electrocompetent and transformed with pKHdbpBAko as described by Yang et al [28]. After electroporation with pKHdbpBAko, spirochetes were allowed to recover overnight at 35°C without antibiotic selection in 20 ml BSK-H. The cells were diluted in BSK-H medium containing the appropriate concentration of antibiotic(s) and aliquoted in 96-well tissue culture plates (Corning, Lowell, MA). Transformants were recovered 7-21 days after plating from wells in which a red to orange color change of the medium was observed. The presence of viable spirochetes was confirmed visually by dark-field microscopy and clones expanded into BSK medium supplemented with kanamycin.
Transformants were verified as dbpBA-deficient mutants by diagnostic PCR using primers ko5 and ko4 (Table 3) followed by analytical restriction enzyme digestion of the PCR product with AscI. DNA for PCR analysis was extracted from borreliae harvested from the expansion cultures. The PCR product generated from amplification of Bb297 DNA (wild-type) is of similar size to that generated by amplification of DNA from a dbpBA::PflgB-kan mutant, but the former lacks AscI sites. Therefore, to verify that the dbpBA operon was replaced by the PflgB-kan cassette, PCR products from both Bb297 and mutant-derived were digested with AscI prior to agarose-gel electrophoresis. The presence of the vlsE and pncA (BBE22) genes in kanamycin-resistant transformants was confirmed by PCR using vlsE-5' and vlsE-3' for vlsE amplification or BBE22-5' and BBE22-3' for BBE22 amplification; refer to Table 3 for sequence information. FlaB5' and FlaB3' primers, which amplified the flaB gene of B. burgdorferi, were used as a control for DNA integrity. A single dbpBA-deficient clone, BbKH500, that retained vlsE and pncA, was chosen for additional PCR-based analyses to compare the endogenous plasmid content of this clone to Bb297. The sequences of the primers utilized for plasmid profiling are provided in Table 4. Nine of the primer pairs have been previously described by Eggers et al. [55]. The remaining primers utilized are unique from those cited by Eggers et al. and were designed primarily based on sequence data from strain 297.

Construction of shuttle plasmids for genetic complementation of BbKH500 with DbpA
Complementation of DbpA in BbKH500 was achieved by transforming electrocompetent BbKH500 with the B. burgdorferi-shuttle plasmid, pKH2000. Because dbpB precedes dbpA in the native operon, it first was necessary to clone the promoter for the dbpBA operon directly in front of the dbpA gene from Bb297, thereby removing the dbpB gene. The dbpBA promoter region (Prom) was amplified by PCR using primers comp1 and comp2 and the dbpA open reading frame (ORF) was amplified using primer comp3 and comp4. To facilitate fusion of the Prom and dbpA open reading frame fragment, an NdeI restriction site was introduced into the 3' end of oligonucleotide comp2 and the 5' end of primer comp3. Following PCR amplification, the resulting PCR fragments were digested with NdeI and ligated together. A second PCR amplification was performed using the ligation product as the template and the oligonucleotides comp1 and comp4 for primers. The resultant PCR product was digested with BglII (a unique BglII site is located in the PCR product, 11 bp downstream of the 3' end of the comp1 primer) and SphI (5' end of comp4 primer) then cloned into the BglII and SphI sites of pJD51, a derivative of pJD44 [27], that contains the aadA gene encoding streptomycin resistance in B. burgdorferi [56], to create pKH2000. Electroporation of BbKH500 was performed as described above and transformants were selected in the presence of kanamycin and streptomycin. Antibiotic-resistant clones first were checked for the presence of the Prom-dbpA construct by PCR using primers ko5 and comp5 (the latter anneals near the middle of the dbpA coding strand); Bb297 = 1.2 kb product; BbKH500 = no product; Prom-dbpA = 535 bp product. Next, DbpA expression in clones identified by PCR confirmation was assessed by immunoblot analysis as described below. Clones that expressed DbpA from the Prom-dbpA construct were assessed for the presence of the vlsE and BBE22 genes by PCR amplification using the primers described above. One clone, BbKH501, was chosen for further characterization. PCR-based plasmid profiling was performed on BbKH501, as described above, to compare the plasmid content of this clone to BbKH500 and Bb297.

Proteinase K digestion of B. burgdorferi
Intact, motile borreliae were exposed to 200 μg of proteinase K (40 mg/ml; Fisher Scientific, Pittsburgh, PA) or were sham treated for 40 min at room temperature. To stop the reaction, 10 μl of phenylmethylsulfonyl fluoride (50 mg/ml in isopropanol; Sigma) was added to each sample and the bacteria prepared for SDS-polyacrylamide gel electrophoresis (PAGE) and immunoblot analysis as described below. Nitrocellulose membranes were probed with either 6B3-DbpA, a mouse monoclonal antibody that specifically recognizes DbpA, chicken anti-B. burgdorferi FlaB IgY, or rat anti-DbpB anti-sera. The monoclonal antibody, 6B3-DbpA, was produced in collaboration with the Antibody Production Core facility at UT Southwestern and the pol-yclonal rat anti-DbpB antisera was described previously [16]. The chicken anti-FlaB antibody was produced in collaboration with Lampire Biological Laboratories (Pipersville, PA). Secondary antibodies were horseradish peroxidase-conjugated goat anti-mouse immunoglobulin G (IgG), donkey anti-chicken IgY, or goat anti-rat IgG (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA) diluted 1:10,000-1:30,000 for chemiluminescent detection. Immunoblots were developed using Immobilon Chemiluminescent Western HRP Substrate (Millipore, Billerica, MA) and exposed to X-ray film (Phenix Research Products, Hayward, CA).

Infection of mice by needle inoculation
Prior to use of the cultures in needle-inoculation experiments, the bacterial cell density in each culture was accurately determined by counting spirochetes in no fewer than 60 microscopic fields (400× magnification) using dark-field microscopy. Cultures exhibiting cell aggregation were not used for infections as the presence of clumps prevented accurate enumeration of spirochete density. Cultures with spirochetes exhibiting reduced motility also were not used for mouse infections. Three-to five-weekold female C3H/HeJ mice (The Jackson Laboratory, Bar Harbor, ME) were used for all studies. Mice were infected via intradermal injection with serial dilutions of B. burgdorferi in BSK medium as previously described [16]. At the appropriate time intervals, ear-punch biopsies were harvested using a 2 mm ear punch, placed in BSK-H medium supplemented with BAM, and cultures were examined by dark-field microscopy for the presence of spirochetes. Aliquots of each culture were passed to media containing antibiotics to confirm the antibiotic-resistance phenotype of the bacteria that were recovered from cultures of the ear punch biopsies. The ID 50 was calculated using the method described by Reed and Muench [57]. UT Southwestern is accredited by the International Association for Assessment and Accreditation of Laboratory Animals Care (AAALAC) and all animal protocols were approved by the Institutional Animal Care and Use Committee (IACUC) at UT Southwestern.

Colonization of Ixodes scapularis larvae with B. burgdorferi
Female C3H/HeJ mice were needle inoculated as described above with either Bb297 (10 4 spirochetes) or BbKH500 (100 μl of a post-exponential growth phase culture; 10 6 -10 7 spirochetes). Infection of mice was confirmed by ear-punch biopsy culture; 4-and 8-weeks postinoculation for Bb297 and BbKH500, respectively. Naïve, pathogen-free I. scapularis larvae, obtained from the Department of Entomology and Plant Pathology at Oklahoma State University (Stillwater, OK), were allowed to feed to repletion on the infected mice individually housed in cages with raised wire-bottoms above water to facilitate recovery of the ticks. Fed larvae were collected and washed sequentially with 70% ethanol, deionized water (dH 2 O), 1× Fungizone (Gemini Bio-Products, West Sacramento, CA), and dH 2 O before placing them in 100% cotton fabric-lined Petri dish molting chambers for storage until they molted to the nymphal stage. The molting chambers containing the ticks were housed in a humidified chamber (97-98% humidity) containing saturated potassium sulfate solution at 20°C with a 16 h light, 8 h dark cycle. Unfed nymphs were collected and stored in autoclaved glass vials containing approximately 1 cm of sand. The vials were closed with vented lids and the ticks housed as described above.

Direct immunofluorescence assay (IFA) on B. burgdorferiinfected ticks
Prior to placement of unfed (flat) nymphs on naïve mice, five nymphs from each of two individual mice were dissected on silylated slides (CEL Associates, Inc., Pearland, TX) in 50 μl phosphate-buffered saline containing 10 mM MgCl 2 . At the time testing was performed, approximately 11 weeks had elapsed since these ticks had fed to repletion as larvae. Midgut tissues were extracted from the ticks, allowed to dry on the slides, blocked with Tris-buffered saline with 0.1% Tween-20, then probed with a FITC-conjugated rabbit anti-B. burgdorferi antibody (Fitzgerald Industries International, Inc., Concord, MA) as described previously [27].

Transmission of B. burgdorferi from infected nymphal ticks to naïve mice
To assess the transmissibility of strain Bb297 and BbKH500 from flat nymphs to naïve mice, either five or ten ticks were allowed to feed to repletion on three-to five-week-old female C3H/HeJ mice. At three-weeks postinfestation, ear-punch biopsies were harvested from each mouse and cultured in BSK-H medium without antibiotics to determine infection status. Borreliae from the earpunch biopsy cultures were transferred to BSK-H medium supplemented with kanamycin and streptomycin to confirm their antibiotic-resistance phenotypes.

Statistical analysis
Statistical analysis was performed with assistance from the UT Southwestern Clinical Sciences Department. Both chisquare and Fisher's exact test were applied to the mouse infection data in pair-wise comparisons between experimental groups.


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Flow-Cytometric Phosphoprotein Analysis Reveals Agonist and Temporal Differences in Responses of Murine Hematopoietic Stem/Progenitor Cells

Hematopoietic stem cells (HSCs) are probably the best-studied adult tissue-restricted stem cells. Although methods for flow cytometric detection of phosphoproteins in hematopoeitic progenitors and mature cells are available, analogous protocols for HSC are lacking. We present a robust method to study intracellular signaling in immunophenotypically-defined murine HSC/progenitor cell (HPC)-enriched populations. Using this method, we uncover differences in the response dynamics of several phosphoproteins representative of the Ras/MAP-Kinase(K), PI3K, mTOR and Jak/STAT pathways in HSC/HPCs stimulated by Scf, Thpo, as well as several other important HSC/HPC agonists.

Much progress has been made in determining the physiological function of HSCs/HPCs. Less is known about their biochemical responses to various agonists, largely because traditional approaches (e.g., immunoblotting) are not applicable to such rare cells. Recently, murine HSC purified by Fluorescence-Activated Cell Sorting (FACS) were stimulated ex vivo, and changes in their phosphoprotein levels were visualized by immunofluoresence [2]. Although this approach provides some insights into HSC signaling and also permits the analysis of protein subcellular localization, it only allows analysis of one cellular population at a time and is subject to the limitations of immunofluorescence (e.g., difficulties in quantification, photobleaching, etc.). BM cells also have been stimulated ex vivo, then fixed, permeabilized, immunostained for surface and intracellular markers, and analyzed by FACS [3]. However, this method only permits analysis of the highly heterogeneous LIN 2 Kit + population. There is one report of intracellular staining of LIN 2 cells that were first surface-stained for Sca-1 and Kit, and then fixed and saponin-permeabilized with a commercial kit [4]. However, this permeabilization agent may not be optimal for HSC/HPC signaling studies (see below).
We have optimized conditions to analyze signal transduction pathways in phenotypically-defined HSC/HPC subsets. We focused on two well-characterized agonists, stem cell factor (Scf) and thrombopoietin (Thpo), as well as several other important HSC/HPC growth factors and cytokines, and demonstrate robust responses in several well-studied signaling pathways. Using our approach we observed signaling cross-talk between the MEK and mammalian target of rapamycin (mTOR) pathways, converging on ribosomal protein S6 phosphorylation in HSC/HPC.

Fixation conditions for retaining surface antigenicity of sorted Lin 2 cells
We sought a robust protocol for multi-parametric, quantitative analysis of intracellular signaling in phenotypically-defined murine stem/progenitor cell populations [5][6][7]. Previous studies showed that many extracellular proteins maintain sufficient antigenicity for FACS after paraformaldehyde (PFA) fixation and alcohol-based permeabilizaton [5], and established that alcohol-(or ketone-) based permeabilization is superior to detergents for preserving intracellular phosphoproteins [6]. A major barrier to applying these methods to HSCs/HPCs arises from the requirement that multiple lineage markers be used to enrich for the LIN 2 HSC/ HPC compartment (Materials and Methods). Some important extracellular antigens lose antigenicity after fixation/permeabilization [5], and contamination of the LIN 2 compartment with LIN + cells rendered artifactually ''LIN 2 '' by fixation/permeabilization could seriously confound analysis. We decided to enrich for HSC/ HPC by first removing LIN + and dead cells (propidium iodide [PI] + ) by FACS ( Figure 1A and B). We avoided positive selection with HSC/HPC-specific antigens to limit potential antibodytriggered signaling events [8]. LIN 2 cells represent a minority of murine BM (,15%), and we obtained this population at reasonably high purity with an average yield of 5.75610 5 (6) 2.38610 5 cells/mouse (mean6standard deviation [SD]; n = 13 independent experiments; Figure 1B). A minimum of 1610 5 LIN 2 PI 2 cells, comprising ,100 LSK and .200 LK cells after fixation/permeabilization, is sufficient for analysis of signaling events following brief ex vivo culture and agonist stimulation ( Figure 1A and data not shown).
We next sought conditions that preserve surface antigens on HSC/HPC. Consistent with previous reports [3,5], Kit staining was maintained after PFA fixation and permeabilization with multiple agents (Figure 1C and S1). However, Sca-1 antigenicity was destroyed after PFA fixation and methanol permeabilization [3], and significantly reduced following PFA fixation and permabilization with either ethanol, methanol, isopropranol, Triton (0.50%), or two concentrations of saponin. Saponin permeabilization also increased non-specific binding ( Figure S1). There was considerable retention of Sca-1 antigenicity on LIN 2 Kit + cells following acetone or 0.10% Triton treatment ( Figure 1C and S1), although the median fluorescent intensity (MFI) of these cells relative to untreated or PFA-fixed cells was reduced. After adjusting the gating to account for the reduced MFI, a distinct population of LSK cells could still be identified. Acetone was superior to Triton in preserving Sca-1 antigenicity (as indicated by the significantly higher MFI) in the LIN 2 Kit + population ( Figure S1), and was reported previously to provide superior preservation of intracellular phosphoprotein epitopes (compared to detergents) [6]; thus, we used acetone in all subsequent experiments.
Following Scf treatment, robust pERK and pS6 responses were observed in LSK and LK, but not LDN cells (Figure 2A; quantified in Figure S3). The pERK response was specific, as it was eliminated by pre-treating cells with the MEK inhibitor UO126 (Figure 2A). S6 can be phosphorylated on Ser235/236 as a consequence of mTOR or ERK activation [15]. Scf-evoked phosphorylation of these residues at 5 min in either LSK or LK cells was primarily UO126-, not rapamycin-sensitive, and therefore MEK, rather than mTOR-dependent ( Figure 2A). We also observed a small, PI3K-dependent response, in pAKT ( Figure 2A). We failed to detect significant Scf-evoked increases in STAT3 or STAT5 phosphorylation in any of the cell populations examined (Figure 2A). Although this could indicate a lower sensitivity for detecting pSTAT than other p-epitopes, phosphorylation of both STAT proteins was detected in response to Thpo, as well as several other agonists (see below).
Thpo also induced a strong increase in pERK and pS6 in LSK cells. Unlike Scf, which affected LSK and LK populations equally, Thpo-evoked responses were lower in LK cells ( Figure 2A). Thpo also evoked a small, but specific, increase in pAKT ( Figure 2A). Unlike Scf, Thpo induced significant increases in pSTAT5 and pSTAT3 in LSK cells and in pSTAT5 alone in LK cells ( Figure 2A). Consistent with our findings, Thpo-evoked increases in pAKT, pSTAT5 and pSTAT3 were observed previously in LSK CD34 2 cells using immunofluorescence-based assays [2,16]. Treatment with either Scf or Thpo and/or various kinase inhibitors had no effect on surface marker antigenicity in either cell population ( Figure S4).
We also assessed phosphoprotein responses to other agonists commonly used ex vivo in HSC/HPC assays and culture conditions. Flt3 ligand (Flt3L) evoked strong increases in pERK and pS6 in most LSK cells and a minority of LK cells, and a smaller response in pAKT levels in LSK cells ( Figure 2B; quantified in Figure S5). Like Scf, also a receptor tyrosine kinase agonist, Flt3L failed to evoke detectable changes in STAT phosphorylation ( Figure 2B). Interleukin-3 (IL-3) elicited stronger responses in LK cells than in LSK cells, and increased pERK, pS6, and to a lesser extent pSTAT5 ( Figure 2B and S5). Granulocyte/ macrophage colony-stimulating factor (Gm-csf) evoked small increases in pERK, pS6 and pSTAT5, predominantly in LK cells, consistent with Gm-csf receptor levels being detected in GMP [17] ( Figure 2B and S5). In both LSK and LK cells, IL-6 only evoked STAT3 phosphorylation with no changes in any of the other phosphoproteins analyzed ( Figure 2B and S5).
Using CD34 staining, we also analyzed signaling responses in more refined LSK/LK subsets (Figure 3, S6, and S7). Scf evoked ''Primary Sort'' indicates typical gates for LIN 2 /PI 2 cells after debris and cell clumps were gated using forward scatter/side scatter (FSC/SSC). ''Re-analysis'' represents cells analyzed for LIN/PI staining immediately after sorting. Numbers indicate percentage (6) standard deviation (SD) (n = 5) of LIN/PI-stained BM cells before and after sorting. C, Cells were sorted as in (B), incubated in vitro at 37uC for ,1 hour in 2% FBS/IMDM, and either left untreated, fixed with 1.5% PFA without permeabilization, or fixed with PFA and subsequently permeabilized with ice-cold ,100% acetone. Cells were then stained with Kit (APC-conjugate) and Sca-1 (PE-conjugate) antibodies and analyzed with a flow cytometer. Shown are pseudocolor dot plots and gating schemes, along with staining of BM from BALB/c mice, which express low levels of Sca-1 [9]. The percentage of the parental gate 6SD from 4 independent experiments is indicated. LK, LIN 2 Kit + Sca-1 2 , LSK, LIN 2 Kit + ,Sca-1 + , LDN, LIN 2 Kit 2 Sca-1 2 . A minimum of 2,500 events was collected (not counting cells with high SSC and low FSC, gated as in panel (B), and hereafter, termed ''FSC/SSC-gated events''). D, LIN 2 /PI 2 cells were left untreated or fixed, permeabilized, and stained for Sca-1 and Kit (as above), as well as CD34 (Pacific Blue-conjugate). Shown are pseudocolor dot plots, contour plots, and the percentage of the parental gates (from one of several experiments). A minimum of 1,500 FSC/SSC-gated events was collected. CD34 gates were set according to fluorescence minus one controls (data not shown). LT, long-term HSC, MPP, multipotent progenitors, GMP, granulocyte/monocyte progenitors, CMP, common-myeloid progenitors, MEP, megakaryocyte/ erythroid progenitors. doi:10.1371/journal.pone.0003776.g001 Figure 3. Detection of phosphoproteins in CD34-defined LSK/LK subsets. A, Cells were treated as in Figure 2A and gated for CD34 expression and pERK (Thr202/Tyr204) levels. Contour plots and percentage of parental gates are shown from a representative experiment performed at least twice. Minima of 6,000, 4,700, and 2,700 FSC/SSC-gated events were collected for the Scf, Thpo, and other agonist experiments, respectively. Using CD34-gated HSC/HPC subsets permit analysis of signaling events in more discretely defined HSC/HPC populations. B, Cells were treated with Thpo for 5 minutes, processed as described above, and stained simultaneously for surface markers, pS6 (Ser235/236), and pSTAT5 (Tyr694) in the indicated LSK and LK subsets (gated as in Figure 1D). Data are representative of 2 independent experiments, with a minimum of 7,400 FSC/SSC-gated events collected. Note the ability to simultaneously detect multiple phospho-epitopes in defined HSC/HPC subsets. doi:10.1371/journal.pone.0003776.g003 nearly uniform ERK phosphorylation in LSK and LK subsets. In contrast, Thpo responses were more heterogeneous. Most LSK cells increased pERK levels in response to Thpo, yet a substantial minority of CD34 + /CD34 2 LSK cells did not respond ( Figure 3A). The response to Thpo in LK cells was also heterogeneous, with only minor fractions of CD34-positive and -negative cells showing activation ( Figure 3A). These findings are consistent with expression of c-Mpl on CD34 + CMPs and CD34 2 MEPs [11]. Flt3L also evoked a heterogeneous response in LSK and LK cells. In both, LSK and LK cells, elevated Flt3L-evoked pERK levels were mostly detected in CD34 + cells, although some of these cells showed no increase in pERK ( Figure 3A and S2). IL-3-evoked phosphorylation of ERK was equivalent in CD34 + and CD34 2 LSK cells, but was stronger in CD34 + LK cells (as opposed to CD34 2 cells). In contrast, Gm-csf responses were more apparent in LK cells, with a subset of CD34 + LK cells responding ( Figure 3A) [17]. Other phosphoprotein signaling responses measured in LSK/LK CD34 subsets are presented in Figure S6 and S7. Finally, by using antibodies to pS6 and pSTAT5, we found that several phosphoproteins can be quantified simultaneously in single cells from all of the LSK and LK CD34 subsets tested ( Figure 3B).

Kinetics of signaling events in HSC/HPC
Our initial analyses were performed to assess the selectivity and sensitivity of our protocol and focused on an early time point (5 minutes), which would presumably reveal strong responses and be less influenced by pathway cross-talk. Subsequently, we characterized later responses in several phosphoproteins. Scfevoked ERK phosphorylation (Thr202/Tyr204) was elevated 5 minutes post-stimulation, rapidly declined by 15 minutes and remained at baseline levels for at least 30 minutes post-stimulation in LSK and LK cells ( Figure 4A and data not shown). We observed similar kinetics of response in pERK in response to Thpo (data not shown). Scf-and Thpo-evoked S6 phosphorylation typically peaked by 5 minutes post-stimulation in both LSK and LK cells, declined gradually starting at 15 minutes, and returned to near-baseline levels at 30 minutes for Thpo and 1 hour for Scf ( Figure 4B). Scf-evoked pS6 responses were significantly higher than Thpo-evoked responses in LSK and LK cells throughout the time course ( Figure 4B). The combination of Scf and Thpo did not lead to an enhanced pS6 response relative to Scf treatment at most time points in either LSK or LK cells ( Figure 4B). However, S6 phosphorylation was sustained significantly in LSK, but not LK cells, when both agonists were used compared to either agonist alone ( Figure 4B; Fold change in MFI at 60 minutes with Scf+Thpo: 7.0762.14; Scf alone: 1.6260.13 p = 0.04; Thpo alone: 1.4760.15, p = 0.04).
As noted above (Figure 2 and S3), pSTAT5 levels were higher in Thpo-stimulated LSK cells, compared to Scf-stimulated cells. These levels appeared to peak at 15 minutes post-Thpo stimulation, and remained elevated slightly even at 60 minutes in LSK cells ( Figure 4B). Scf did not affect STAT5 phosphorylation appreciably over this time course either alone or in combination with Thpo treatment ( Figure 4B).

Differential control of S6 phosphorylation at Ser235/236 in HSC/HPC
Scf-evoked phosphorylation of ribosomal protein S6 at Ser235/ 236 at 5 minutes was completely sensitive to MEK inhibition and insensitive to mTOR inhibition (Figure 2A and S3). However, S6 phosphorylation remained elevated at 15 minutes post-Scf addition, whereas pERK levels declined rapidly after 5 minutes ( Figure 4A and B). At 15 minutes post-Scf addition, pS6 levels were both MEK-and mTOR-dependent in LSK and LK cells, as the fold change in MFI for pS6 was reduced by either UO126 or rapamycin pre-treatment. These data are similar to previously reported results on mouse and human cell lines [15,18], and provide proof of principle for the utility in pathway analysis of our phosphoflow protocol in HSC/HPC-enriched populations.

Discussion
We have developed a protocol that utilizes the multi-parametric and quantitative attributes of FACS to examine multiple phosphorylation events at the single cell level in highly purified HSC/HPC populations. Previous studies have only been able to analyze signaling events in bulk hematopoietic progenitors by flow cytometry [3] or one cellular parameter at a time by immunofluorescence-based techniques [2]. Although still limited by low target cell numbers and loss of a significant proportion of the starting material post fixation/permeabilization and washing steps, our protocol permits the simultaneous analysis of multiple cellular (HSC, MPP, GMP/CMP, MEP), and intracellular parameters in HSC/HPC, akin to studies of more mature hematopoietic cells [6].
Intracellular signaling networks in HSC/HPC have remained largely unexplored territory, mostly due to the rarity of these cell populations. Here we have analyzed signal transduction responses to several HSC/HPC agonists, including Scf and Thpo, both of which are important in vivo for HSC function and can promote survival and enhance the proliferation of HSC/HPC ex vivo [2,[19][20][21][22][23]. Although Scf elicited rapid responses in some intracellular pathways that we interrogated (Ras/MAPK, PI3K/AKT) in all of the HSC/HPC cellular subsets we analyzed, Thpo stimulation profiles were stronger in HSC/MPP (compared to GMP/CMP or MEP) and consisted of the activation of a broader array of intracellular pathways (Ras/MAPK, PI3K/AKT, JAK/STAT). Other agonists, such as Flt3L, appeared to elicit stronger responses in LSK compared to LK cells, whereas Gm-csf elicited stronger responses in LK cells. These differences in response could reflect a heterogeneous expression of receptors in LSK and LK cells, as well as differences in the intrinsic sensitivity to these agonists between populations. We detected a significant synergistic effect of Scf and Thpo relative to either treatment alone on pS6 levels in LSK, resulting in a sustained pS6 response ( Figure 4B). It is tempting to speculate that the reason for biological synergy (in terms of colony formation or proliferation/survival of HSC/HPC) previously observed between Scf and Thpo [2,[19][20][21] reflects sustained Ras/MAPK and mTOR signaling, although further genetic-or pharmacology-based biological assays are required to explore this point. Recent studies have demonstrated that both Thpo and Kit are required to maintain the steady-state pool of adult quiescent HSC [22,24]. While STAT5 plays a significant role in HSC function [16,25,26], the role of members of the Ras/ MAPK or mTOR pathway in HSC biology remains largely unexplored. Our data suggest that these pathways likely play a role downstream of agonist stimulation in HSC/HPC.
We also simultaneously assessed responses in pS6/pSTAT5 in CD34-LSK and LK subsets ( Figure 3B). In all populations analyzed, approximately half of the cells respond to Thpo stimulation by elevating both pS6 and pSTAT5, but the rest only elevate pS6 ( Figure 3B). This could reflect the relative hypersensitivity of the pathways downstream of Thpo leading to S6 phosphorylation over signaling to STAT5, lower sensitivity of our conditions for detecting pSTAT5, or intrinsic heterogeneity in the cell types present in these compartments. Regardless, the physiological relevance of this heterogeneity in signaling responses merits further investigation. Our approach can be used along with selective inhibitors to examine pathway-dependencies. For example, our analysis of Scfevoked S6 phosphorylation in primary HSC/HPC is largely in agreement with that of Roux et al. [15], who demonstrated that serum-evoked S6 phosphorylation at Ser235/236 in human cell lines (HEK 293) is dependent on an early MEK-dependent pathway (through RSK) and a later MEK-and mTOR-dependent pathway. Our data are also consistent with the Mek-inhibitor sensitivity of Ser235/236 phospohorylation in mouse embryonic fibroblasts doubly deficient for the mTOR downstream kinases S6K1/2 [18]. Our results suggest a model in which Scf stimulation leads to ERK activation that likely leads to RSK1/2 activation and S6 phosphorylation at Ser235/236 at 5 minutes post stimulation. At 15 minutes, S6 phosphorylation, is still partially UO126-and rapamycin-sensitive, although levels of pERK have returned to baseline at this time point. ERK activation also can lead to TSC2 phosphorylation and inactivation [27]. Our data suggests the intriguing possibility that ERK mediated TSC2 inactivation is required along with mTOR-dependent phosphorylation of S6 to sustain high levels of pS6 in HSC/HPC. Direct testing of this model will be possible with the availability of appropriate phosphospecific antibodies.
Finally, our protocol should be amenable to the study of signal transduction abnormalities in leukemia stem cells (LSC), as these cells often share phenotypic similarities with normal HSC/HPC [17,28]. The effects of drugs that may affect deregulated signal transduction pathways in leukemia can be readily tested in cell populations expressing surface epitopes examined in this study.

Cell preparation and FACS
BM was harvested from both limbs of 8-12-week-old C57BL/6 or BALB/c mice (Charles River Laboratories, Wilmington, MA) euthanized by CO 2 asphyxiation, followed by cervical dislocation, flushed into cold 2% FBS/IMDM and passed through a 40 mm cell strainer (BD Falcon). Cells were collected by centrifugation, and red blood cells were lysed in 0.16 M NH 4 Cl on ice. Cells were then washed, centrifuged, resuspended in 400 ml of cold 2% FBS/ IMDM, and stained with saturating levels of antibodies against the above lineage markers for ,15 minutes on ice. After washing, cells were passed through a 40 mm cell strainer into 5-ml polypropylene round-bottom tubes (BD), and stained with 1 mg/ml PI (Invitrogen, Carlsbad, CA). LIN 2 /PI 2 cells were purified using a FACSAria (BD). All mouse studies were approved by our Institutional Animal Care and Use Committee.

Statistical analysis
The statistical significance of differences between population means was assessed by 2-tailed unpaired Student t test.

Supporting Information
Materials and Methods S1